The advent and widespread application of the recombinant DNA methodology during the past twelve years has greatly extended our understanding of the molecular basis of heredity. More significantly, the emergence of a new molecular paradigm - reverse genetics, which relies on the ability to specifically modify the structure of genes in vitro and to test their biological properties, has revolutionized the experimental approaches in molecular genetics. The ability to construct minichromosomes with novel arrangements of genetic elements also provides vectors for the isolation of genes as well as substrates for the analysis of specific genetic reactions. Our proposal, which applies this approach to explore the basic mechanisms of gene regulation in the establishment of immune responsiveness and of genetic recombination, aims (1) identify the factors and mechanisms that limit the epression of antibody genes to only lymphoid cells; (2) isolate and characterize the human gene whose dysfunction is responsible for galactosemia; (3) understand the mechanisms responsible for non- homologous, homologous and non-reciprocal modes of genetic recombination, especially those which permit the rejoining of broken DNA; (4) develop genetic approaches to effect a replacement or repair of chromosomal DNA sequences; (5) developing vectors which can deliver genes to only specific types of cells. It is anticipated that the information we develop will advance our unerstanding of the molecular basis of disease and provide novel approaches to their prevention, treatment and cure.